| Objectives:The growth and metabolism of bone are controlled by osteoblasts（osteogenesis）and osteoclasts（absorption）,and osteoblasts play a leading role in the process of bone formation.The mechanical environment is an important factor affecting bone remodeling.Osteoblasts are important effector cells for bone response to mechanical stimulation.However,mechanical stimulation affects the biological processes such as proliferation,differentiation,and mineralization of osteoblasts.Factors such as intensity,manner,and duration can change the biological characteristics of osteoblasts,and thus affect the functional activity of osteoblasts.Various non-war military operations such as high-intensity operations,sports,and disaster relief make people in an overloaded mechanical environment.At present,although related studies have investigated the effects of overloaded mechanical stimulation on osteoblasts,the specific effect mechanism remains unclear.In recent years,a large number of experimental studies have demonstrated that icariin can promote the proliferation,differentiation and mineralization of osteoblasts,and icariin at a certain concentration of drugs plays an important role in the repair of bone damage,and studies of icariin on overload In this study,a two-dimensional overload injury model of osteoblasts was constructed using a four-point bending mechanics loading device to study the effects of mechanical overload stimulation on the biological characteristics of osteoblasts such as proliferation,differentiation,apoptosis,and mineralization.The response mechanism of bone cells to mechanical overload stimulation.To study the effect of icariin on overloaded osteoblasts and to reveal the preventive and therapeutic mechanism of icariin for overload bone injury.To provide a new research method for the study of bone injury repair and reconstruction under overload mechanical stimulation,and then provide a theoretical basis for clinical treatment.Methods:1.Icariin at concentration of 0,10-4,10-5,10-6,10-7,10-8,10-9mol/L was used to interfere the mouse osteoblastic cell line MC3T3-E1.The proliferation of osteoblasts was evaluated by MTT method.The cell density and morphology were observed by HE staining.The alkaline phosphatase kit was used to evaluate the differentiation of osteoblasts.Alizarin red staining was used to detect the mineralization ability of osteoblasts.Finally,the optimal concentration of icariin to enhance the proliferation,differentiation and mineralization of MC3T3-E1 cells was screened from each group of different drug concentrations.2.Two overload damage models were constructed using a four-point bending mechanics loading device.The applied mechanical stimuli parameters are the mechanical tensile strain with the frequency of 1.5Hz,the intensity of 3000με,the daily three-day period of 6h and the frequency of 0.5Hz,the mechanical tensile strain of the strength of 5000με,and the daily tension of 0.5h for three consecutive days.After overload mechanics stimulation,the proliferation of osteoblasts was detected by MTT assay;the apoptosis was detected by flow cytometry;the cytoskeleton was evaluated by confocal microscopy;RT-PCR And Western Blot detection of cell differentiation marker gene expression;alizarin red staining cell mineralization.3.The optimal concentrations of icariin was added to the cultured MC3T3-E1cells before and after overloaded mechanical stimulation,and the proliferation,apoptosis,differentiation,and mineralization of osteoblasts were detected after the addition of the drug.4.RT-PCR and Western Blot methods were used to detect the effects of icariin against overload mechanical stimulation on the expression of each molecule in Wnt/β-catenin signaling pathway.Immunofluorescence was used to detect the effect of icariin on the activation ofβ-catenin protein and its migration to the nucleus.Results:1.Icariin can promote the proliferation of osteoblasts.Among them,10-7mol/L icariin significantly promoted the proliferation of osteoblasts.HE staining suggested that osteoblasts generally have an irregular morphology,such as triangular,long spindle,and polygonal shapes.Among them,the experimental group had the largest number of osteoblasts and the highest cell density at the drug concentration of10-7mol/L.Icariin can significantly increase the activity of alkaline phosphatase in osteoblasts.Among them,osteoblasts with a concentration of 10-7mol/L had the highest alkaline phosphatase activity;10-4mol/L group had the lowest alkaline phosphatase activity.Alkaline phosphatase activity in the 10-9mol/L experimental group was slightly higher than that in the blank control group,but the difference was not significant.A certain concentration of icariin can improve the osteoblast mineralization ability.A large number of calcium nodules can be seen in the icariin group at the concentration of 10-7mol/L.2.Compared with the blank control group,the mechanical overload group significantly inhibited the proliferation activity of osteoblasts;compared with the control group,the mechanical overload group obviously promoted the apoptosis of osteoblasts;Under laser confocal microscope observation,the osteoblasts in the mechanical overload group shrank and deformed,the arrangement of microfilaments was disordered,the skeleton arrangement was loose,the outline was blurred,the orientation was poor,and the skeleton even appeared broken.No obvious changes in the nucleus were observed.The results of RT-PCR and Western Blot showed that mechanical overload stimulated the differentiation of osteoblast differentiation marker genes and protein expression.The results of alizarin red staining showed that mechanical overload could inhibit the formation of osteoblasts calcium nodules.3.Compared with the mechanical overload group,the icariin intervention group significantly promoted the proliferation activity of osteoblasts,and the prophylactic activity of the prophylactic group was higher than that of the post-injury group.Compared with the mechanical overload group,the apoptosis rate of the icariin intervention group was low,and the apoptosis rate of the prophylactic group was the lowest.Under laser confocal microscope observation,most osteoblasts in the prophylactic group showed typical triangular,long spindle,and polygonal irregular distributions,and it was difficult to see cell shrinkage and disorganized microfilaments.The results of RT-PCR and Western Blot indicated that the intervention of icariin could increase the expression of osteoblast differentiation marker genes and proteins.The results of alizarin red staining showed that the intervention of icariin could effectively increase the formation of calcium nodules in osteoblasts.4.RT-PCR and Western Blot experiments showed that mechanical overload stimulation significantly inhibited the expression ofβ-catenin and Runx2 genes and proteins in the Wnt/β-catenin signaling pathway.The intervention of the drug icariin could increase the expression ofβ-catenin and Runx2 gene and protein after mechanical overload injury,and the prophylactic administration group was higher than that after injury.After immunofluorescence staining,under microscope observation,overload mechanic stimulation significantly inhibited the expression ofβ-catenin protein in Wnt signaling pathway.The intervention of icariin could improve the expression ofβ-catenin protein after mechanical overload injury.Medium post-injury administration.Conclusions:1.Icariin at a certain concentration can promote the proliferation,differentiation and mineralization of osteoblasts.The optimum concentration of Icariin is 10-7mol/L.2.Overloading mechanical stimulation significantly inhibited osteoblast proliferation,differentiation and mineralization.3.Icariin can repair osteoblast damage caused by mechanics overload to a certain extent,and the preventive effect is better than that after injury.4.The anti-overload mechanical stimulant drug icariin promotes the proliferation,differentiation,and mineralization of osteoblasts by affecting the expression of Runx2 protein by affecting the canonical Wnt/β-catenin signaling pathway. |
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